This invention concerns methodology and a reagent system for volume differentiation of at least two populations of leukocytes and, more specifically, the classifying and counting of at least three populations of leukocytes (1) a lymphocyte population, (2) a monocyte population, and (3) a granulocyte population which includes neutrophils, eosinophils and basophils, using the Coulter Counter.RTM. Model S Plus automated blood counter.
Such data are useful as a screening tool for calling attention to abnormal leukocyte ratios. Abnormal situations identified by this method give information of diagnostic significance and alert the technologist to the need for further study.
Separation of normal human leukocytes by volume distribution utilizing the principle of counting and sizing employed in Coulter Counter.RTM. instruments is now applied as a clinical diagnostic method. This method is based on the fundamental property of all living cells to maintain a certain size and shape. Each type of cell in the circulating blood has its own characteristic volume ranging from as small as 3 cubic microns, i.e. 3 fL, for platelets to 650 fL for polymorphonuclear cells. Advanced Coulter Counter.RTM. instruments have been designed to make use of this volume differential for the purposes of counting and determining the size distribution of platelets, leukocytes, and erythrocytes in order to detect and monitor pathological states.
Erythrocytes and the lymphoid leukocytes unfortunately overlap considerably in cell sizes, and it is not practical to count one in the presence of the other by size discrimination alone. Traditional practice involves the use of a strong lytic-surfactant reagent that stromatolyses the erythrocytes, reducing them to very small particles or causing membrane solubilization, and strips the cytoplasm from both the lymphoid and the myeloid leukocytes, leaving only the lyse-resistant nuclei to be counted. Since original cell volume is drastically affected and reduced to a minimum, only a single leukocyte population is visible by the usual size distribution analysis.
The Coulter Counter.RTM. Model S Plus automated blood cell counter is designed to dilute a sample of whole blood in an isotonic diluent, add a lysing agent, and shortly thereafter begin counting. Thus, a diluent-lysing system must provide erythrocyte lysing kinetics sufficiently rapid to effect complete stromatolysation of the red blood cells (erythrocytes) during the lysing period. In addition, changes in leukocyte volume must be minimal during the data collection step, and ideally should be stable for several minutes. The reagent system must also preserve the integrity of the erythrocyte and platelet number and size distribution, the hemoglobin absorbance spectrum and the total leukocyte count. Finger stick bloods must be stable when prediluted in the isotonic diluent for at least two hours.
In U.S. Pat. No. 3,874,852 (1975) to Coulter Diagnostics, Inc., a formula is included for a composition containing quaternary ammonium salt detergent and potassium cyanide to be employed as a lysing and chromagen-forming reagent for obtaining a single volume leukocyte count ahd hemoglobin determination in the Coulter Counter.RTM. Model S.
In U.S. Pat. No. 4,286,963 to Coulter Electronics, Inc., a lytic diluent for the rapid lysing of red blood cells in whole blood for making a differential determination of lymphoid/myeloid populations of leukocytes, and also measuring hemoglobin by chromagen formation, contains a mixture of an aqueous saline solution of at least one quaternary ammonium salt having surface acting properties, and certain additives such as 2-phenoxyethanol.
In U.S. Pat. No. 4,346,018 to Coulter Electronics, Inc., and in continuing application Ser. No. 395,530 filed July 6, 1982, an isotonic multi-purpose blood diluent and a method for use of this diluent with a lysing reagent system are described for the differential two-volume determination of lymphoid-myeloid populations of leukocytes. An important difference between the two applications is that in claim 1 chlorhexidene diacetate is a named ingredient only in U.S. Pat. No. 4,346,018. This ingredient is also omitted in the preferred embodiment of this application.
To achieve an analysis of the three-volume populations of lymphocyte, monocyte and granulocyte cells in the blood, the leukocyte volume histogram needs to show cleanly separated populations, with little cellular debris, allowing valleys very close to the baseline. Integration of each population will give the relative numbers of the lymphocyte, monocyte and granulocyte cells. The lymphoid population has been demonstrated to contain lymphocytes and small atypical lymphocytes, while the myeloid population contains two sub-groups, the monocytes and granulocytes. The granulocytes include neutrophils, eosinophils, and basophils.